Respiratory syncytial virus (RSV) is a Parmixovirus of the Pneumovirus genus which commonly infects the upper and lower respiratory tract. It is so contagious that by age two, a large percentage of children have been infected by it. Moreover, by age four, virtually all humans have an immunity to RSV.
Typically, RSV infections are mild, remaining localized in the upper respiratory tract and causing symptoms similar to a common cold which require no extensive treatment. However, in some subjects, e.g., immunosuppressed individuals such as infants, elderly persons or patients with underlying cardiopulnonary diseases, the virus may penetrate to the lower respiratory tract requiring hospitalization and breathing support. In some of these cases, RSV infection may cause permanent lung damage or even be life threatening. In the United States alone, RSV results in about 90,000 hospitalizations each year, and results in about 4500 deaths.
RSV appears in two major strain subgroups, A and B, primarily based on serological differences associated with the attachment glycoprotein, G. The major surface glycoprotein, i.e., the 90 kD G protein, can differ up to 50% at the amino acid level between isolates Johnson et al, Proc. Natl. Acad. Sci. (1987), 84, 5625-5629. By contrast, a potential therapeutic target, the 70 kD fusion (F) protein, is highly conserved across different RSV strains, about i.e., 89% on the amino acid level Johnson et al, J. Gen. Virol. (1988), 69, 2623-2628, Johnson et al, J. Virol. (1987), 10, 3163-3166, P. L. Collins, Plenum Press, N.Y. (1991), 103-162. Moreover, it is known that antibodies elicited against F-protein of a given type are cross-reactive with the other type.
The F-protein is a heterodimer, generated from a linear precursor, consisting of disulfide-linked fragments of 48 and 23 kD respectively Walsh et al, J. Gen. Virol, (1985), 66, 401-415. Inhibition of syncytia formation by polyclonal antibodies is associated with significant reaction to the 23 kD fragment.
As noted, while RSV infections are usually mild, in some individuals RSV infections may be life threatening. Currently, severe RSV infection is treated by administration of the antiviral agent Ribavarin. However, while Ribavarin exhibits some efficacy in controlling RSV infection, its use is disfavored for several reasons. For example, it is highly expensive and may be administered only in hospitals. Other known RSV treatments only treat the symptoms of RSV infection and include the use of aerosolized bronchodilators in patients with bronchiolitis and corticosteroid therapy in patients with bronchiolitis and RSV pneumonia.
To date, RSV vaccines intended to boost antiviral protective antibodies have been largely unsuccessful. For example, a vaccine based on formalin-inactivated RSV that was tested approximately 25 years ago, induced antibodies that were deficient in fusion inhibiting activity Murphy et al, Clinical Microbiology (1988), 26, 1595-1597, and sometimes even exacerbated the disease. This may potentially be explained to the inability of the formalin inactivated virus to induce protective antibodies. While high antibody titers were measured in vaccine recipients, specific protective titers were lower than in the control population. This may be because formalin inactivated RSV does not display the necessary conformational epitopes required to elicit protective antibodies.
While there is no known effective RSV vaccine to date, there exists some clinical evidence that antibody therapy may confer protection against RSV infection in susceptible individuals, and may even clear an existing RSV infection. For example, it has been reported that newborn infants show a low incidence of severe bronchiolitis, which is hypothesized to be attributable to the presence of protective maternal antibodies Ogilvie et al, J. Med Virol (1981), 7, 263-271. Also, children who are immune to reinfection exhibit statistically higher anti-F-protein titers than those who are reinfected. Moreover, intravenous immune globulin (IVIG) prepared from high titer RSV-immune donors reduces nasal RSV shedding and improves oxygenation Hemming et al, Anti. Viral Agents and Chemotherapy (1987), 31, 1882-1886. Also, recent studies have suggested that the virus can be fought and lung damage prevented by administering RSV-enriched immune globulin (RSVIG) Groothuis et al, The New England J. Med. (1993), 329, 1524-1530, K. McIntosh. The New England J. Med. (1993), 329, 1572-1573, J. R. Groothuis. Antiviral Research, (1994), 23, 1-10, Siber et al, J. Infectious Diseases (1994), 169, 1368-1373, Siber et al, J. Infectious Diseases (1992), 165:456-463.
Similarly, some animal studies suggest that antibody therapy with virus neutralizing antibodies may confer protection against RSV or even clear an existing RSV infection. For example, in vitro neutralizing mouse monoclonal antibodies have been reported to protect mice against infection and also to clear established RSV infections Taylor et al, J. Immunology, (1984), 52, 137-142, Stott et al., xe2x80x9cImmune Responses, Virus Infections and Disease, I.R.L. Press, London (1989), 85-104. Also, monoclonal antibodies to the F-protein of RSV have shown high efficacy in both in vitro and in vivo RSV models Tempest et al, Bio/Technology, (1991), 9, 266-271, Crowe et al, Proc. Natl. Acad. Sci. (1994), 91, 1386-1390, Walsh et al, Infection and Immunity, (1984), 43, 756-758, Barbas III, et al, Proc. Natl. Acad. Sci. (1992), 89, 10164-10168, Walsh, et al, J. Gen. Virol. (1986), 67, 505-513. Antibody concentrations as low as 520-2000 xcexcg/kg body weight have been reported to result in almost instant recovery in animal studies Crowe et al, Proc. Natl. Acad. Sci. (1994), 91, 1386-1390. Moreover, these monoclonal antibodies have been disclosed to neutralize both A and B strains, including laboratory strains and wildtype strains. These antibodies were administered either by injection Groothuis et al, The New England J. Med. (1993), 329, 1524-1530, Siber et al, J. Infectious Diseases (1994), 169, 1368-1373 or by aerosol Crowe et al, Proc. Natl. Acad. Sci. (1994), 91, 1386-1390.
Two different types of potentially therapeutic monoclonal antibodies to the RSV F-protein have been previously described in the literature, humanized murine antibodies Tempest et al, Biol. Technology, (1991) 9, 266-271, or true human antibodies (Fab fragments) Barbas III, et al, Proc. Natl. Acad. Sci. (1992), 89, 10164-10168. Humanized murine antibodies were generated by CDR grafting a cross-strain neutralizing murine anti-F-protein antibody onto a generic human Fc, as well as structural areas of the variable part. The human Fab fragments were produced by combinatorial library technology using human bone marrow cells obtained from an HIV positive donor (immunocompromised). The therapeutic in vivo titers of the humanized and human RSV antibodies were 5 and 2 mg/kg body weight, respectively. It is noted, however, that the humanized antibodies were tested in a syncytia inhibition assay, whereas the human anti-RSV Fab fragments were assayed to determine their virus neutralization activity. Therefore, the results reported with the humanized and human anti-RSV antibodies are not directly comparable.
The Fab fragment generated by the combinatorial library technology were disclosed to be efficient in aerosol. This is probably because of the relatively small size of the molecule. These results are highly encouraging because a major target population for an RSV vaccine is infants. Therefore, aerosol is a particularly desirable mode of administration.
However, notwithstanding the previous published reports of humanized and Fab fragments specific to RSV, there still exists a significant need for improved anti-RSV antibodies having improved therapeutic potential, in particular anti-RSV antibodies which possess high affinity and specificity for the RSV F-protein which effectively neutralize and prevent RSV infection.
Antibody therapy can be subdivided into two principally different activities: (i) passive immunotherapy using intact non-labeled antibodies or labeled antibodies and (ii) active immunotherapy using anti-idiotypes for re-establishment of network balance in autoimmunity.
In passive immunotherapy, naked antibodies are administered to neutralize an antigen or to direct effector functions to targeted membrane associated antigens. Neutralization would be of a lymphokine, a hormone, or an anaphylatoxin, i.e., C5a. Effector functions include complement fixation, macrophage activation and recruitment, and antibody dependent cell mediated cytotoxicity (ADCC). Naked antibodies have been used to treat leukemia Ritz et al, S.F. Blood, (1981), 58, 141-152 and antibodies to GD2 have been used in treatments of neuroblastomas Schulz et al,Cancer Res. (1984), 44:5914 and melanomas Irie et al., Proc. Natl. Acad. Sci., (1986, 83:8694. Also, intravenous immune gamma globulin (IVIG) antibodies with high anti-RSV titers recently were used in experimental trials to treat respiratory distress caused by RSV infection Hemming et al, Anti. Viral Agents and Chemotherapy, (1987), 31, 1882-1886, Groothuis et al, The New England J. Med. (1993), 329, 1524-1530, K. McIntosh, The New England J. Med. (1993), 329, 1572-1573, J. R. Groothuis. Antiviral Research, (1994), 23, 1-10, Siber et al, J. Infectious Diseases (1994), 169, 1368-1373.
The therapeutic efficacy of a monoclonal antibody depends on factors including, e.g., the amount, reactivity, specificity and class of the antibody bound to the antigen. Also, the in vivo half-life of the antibody is a significant therapeutic factor.
Still another factor which may significantly affect the therapeutic potential of antibodies is their species of origin. Currently, monoclonal antibodies used for immunotherapy are almost exclusively of rodent origin Schulz et al, Cancer Res. (1984), 44:5914, Miller et al, Blood (1981), 58, 78-86, Lanzavecchia et al, J. Edp. Med. (1988), 167, 345-352, Sikora et al, Br. Med. Bull. (1984), 40:240, Tsujisaki et al, Cancer Research (1991), 51:2599, largely because the generation of rodent monoclonal antibodies uses well characterized and highly efficient techniques Kxc3x6hler et al, Nature, (1975), 256:495, Galfre et al, Nature, (1977), 266:550. However while rodent monoclonal antibodies possess therapeutic efficacy, they can present restrictions and disadvantages relative to human antibodies. For example, they often induce sub-optimal stimulation of host effector functions (CDCC, ADCC, etc.). Also, murine antibodies may induce human anti-murine antibody (HAMA) responses Schroff et al, Can. Res. (1985, 45:879-885, Shawler et al, J. Immunol. (1985), 135:1530-1535. This may result in shortened antibody half-life Dillman et al Mod. (1986), 5, 73-84, Miller et al, Blood, (1983), 62:988-995 and in some instances may cause toxic side effects such as serum sickness and anaphylaxis.
In some subjects, e.g., heavily immunosuppressed subjects (e.g., patients subjected to heavy chemical or radiation mediated cancer therapy Irie et al, Proc. Natl. Acad. Sci. (1986), 83:8694, Dillman et al, Mod. (1986), 5, 73-84, Koprowski et al, Proc. Natl. Acad. Sci. (1984), 81:216-219), use of murine monoclonal antibodies causes limited negative side effects. By contrast, in patients with normal or hyperactive immune systems, murine antibodies, at least for some disease conditions may exhibit limited efficacy.
In an effort to obviate limitations of murine monoclonal antibodies, recombinant DNA techniques have been applied to produce chimeric antibodies Morrison et al, Proc. Natl. Acad. Sci. (1984), 81:216-219, Boulianne et al, Nature, (1984), 312, 644-646, humanized antibodies by xe2x80x9cCDR graftingxe2x80x9d Riechmann et al, Nature (1984), 332, 323-327 and xe2x80x9cveneeredxe2x80x9d antibodies by substitution of specific surface residues with other amino acids to alleviate or eliminate antigenicity.
However, although such antibodies have been used successfully clinically Gillis et al, J. Immunol. Meth (1989), 25:191, they have proven cumbersome to produce. This is because the understanding of the requirements for optimal antigen recognition and affinity is not yet fully understood. Also, the human framework and the mouse CDR regions often interact sterically with a negative effect on antibody activity. Moreover, such antibodies sometimes still induce strong HAMA responses in patients.
Human antibodies present major advantages over their murine counterparts; they induce optional effector functions, they do not induce HAMA responses and host antigen-specific antibodies may lead to identification of epitopes of therapeutic value that may be too subtle to be recognized by a xenogeneic immune system Lennox et al xe2x80x9cMonoclonal Antibodies in Clinical Medicine. xe2x80x9d London: Academic Press (1982).
While human antibodies are highly desirable, their production is complicated by various factors including ethical considerations, and the fact that conventional methods for producing human antibodies are often inefficient. For example, human subjects cannot generally be adequately immunized with most antigens because of ethical and safety considerations. Consequently, reports of isolation of human monoclonal antibodies with useful affinities, xe2x89xa7108 molar to specific antigens are few McCabe et al, Cancer Research, (1988), 48, 4348-4353. Also, isolation of anti-viral human monoclonal antibodies from donor primed cells has proved to be unwieldy. For example, Gomy reported that only 7 of 14,329 EBV transformed cultures of peripheral blood mononuclear cells (PMBC""s) from HIV positive donors resulted in stable, specific anti-HIV antibody producing cell lines Gorny et al, Proc. Natl. Acad. Sci. (1989), 86:1624-1628.
To date, most human anti-tumor aitibodies have been generated from peripheral blood lymphocytes (PBLS) Irie et al, Br. J. Cancer, (1981), 44:262 or tumor draining lymph node lymphocytes schlom et al, Proc. Natl. Acad. Sci. (1980), 77:6841-6845, Cote et al, Proc. Natl. Acd. Sci. (1983), 80:2026-2030 from cancer patients. However, such antibodies often react with intracellular, and thus therapeutically useless antigens Ho et al, In Hybridoma Technology, Amsterdam (1988), 37-57 or are of the IgM class McCabe et al, Cancer Research (1988), 48, 4348-4353, a class of antibodies with lesser ability to penetrate solid tumors than IgGs. Few of these human antibodies hare moved to clinical trials Drobyski et al, R.C. Transplantation (1991), 51, 1190-1196, suggesting that the rescued antibodies may possess sub-optimal qualities. Moreover, since these approaches exploit the testing donor primed B cells, it is clear that these cells are not an optimal source for rescue of useful monoclonal antibodies.
Recently, generation of human antibodies from primed donors has been improved by stimulation with CD40 resulting in expansion of human B cells Banchereau et al, F. Science (1991), 251:70, Zhani et al, J. Immunol. (1990), 144, 2955-2960, Tohma et al, J. Immunol. (19.91), 146:2544-2552 or by an extra in vitro booster step primer to immortalization Chaudhuri et al, Cancer Supplement (1994), 73, 1098-1104. This principle has been exploited to generate human monoclonal antibodies to Cytomegalovirus, Epstein-Barr Virus (EBV) and Hemophilus influenza with cells from primed donors Steenbakkers et al. Hum. Antibod. Hybridomas (1993), 4, 166-173; Ferrarro et al., Hum. Antibod. Hybridomas (1993), 4, 80-85, Kwekkeboom et al., Immunol. Methods (1993), 160, 117-127, with a significantly higher yield than obtained with other methods (32).
Moreover, to address the limitation of donor priming, immunization and cultivation ex vivo of lymphocytes from healthy donors has been reported. Some success in generating human monoclonal antibodies using ex homine boosting of PBL cells from primed donors has been reportel Maeda et al, Hybridoma (1986), 5:33-41, Kozbor et al, J. Immunol. (1984), 14:23, Duchosal et al, Nature (1992, 355:258-262. The feasibility of immunizing in vitro was first demonstrated in 1967 by Mishell and Dutton Mishell et al, J. Exp. Med (1967), 126:423-442 using murine lymphocytes. In 1973, Hoffman successfully immunized human lymphocytes Hoffman et al, Nature (1973), 243:408-410. Also, successful primary immunizations have been reported with lymphocytes from peripheral blood Luzzati et al, J. Exp. Med. (1975), 144:573:585, Misiti et al J. Exp. Med. (1981), 154:1069-1084, Komatsu et al, Int. Archs. Allergy Appl. Immunol. (1986), 80:431-434, Ohlin et al, C.A.K. Immunology (1989), 68:325 (1989) tonsils Strike et al,J. Immunol. (1978), 132:1789-1803 and spleens, the latter obtained from trauma Ho et al, In Hybridoma Technology, Amsterdam (1988), 37-57, Boerner et al, J. Immunol. (1991), 147:86-95, Ho et al, J. Immunol. (1985), 135:3831-3838, Wasserman et al, J. Immunol. Meth. (1986), 93:275-283, Wasserman et al, J. Immunol. Meth. (1986), 93:275-283, Brams et al, Hum. Antibod. Hybridomas (1993), 4, 47-56, Brams et al, Hum. Antibod. Hybridomas (1993), 4, 57-65 and idiopathic thrombocytopenia purpura (ITP) patients Boerner et al, J. Immunol. (1991), 147:86-95, Brams et al, Hum. Antibod. Hybridomas (1993) 4, 47-56, Brams et al, Hum. Antibod. Hybridomas (1993), 4, 57-65, McRoberts et al, xe2x80x9cIn Vitro Immunization in Hybridoma Technology xe2x80x9d, Elsevier, Amsterdam (1988), 267-275, Lu et al, P. Hybridoma (1993), 12, 381-389.
In vitro immunization offers considerable advantages, e.g., easily reproducible immunizations, lends itself easily to manipulation of antibody class by means of appropriate cultivation and manipulation techniques Chaudhuri et al, Cancer Supplement (1994), 73, 1098-1104. Also, there is evidence that the in vivo tolerance to self-antigens is not prevalent during IVI Boerner et al J. Immunol. (1991), 147:86-95, Brams et al, J. Immunol. Methods (1987), 98:11. Therefore, this technique is potentially applicable for production of antibodies to self-antigens, e.g., tumor markers and receptors involved in autoimmunity.
Several groups have reported the generation of responses to a variety of antigens challenged only in vitro, e.g., tumor associated antigens (TAAs) Boerner et al, J. Immunol. (1991), 147:86-95, Borrebaeck et al, Proc. Natl. Acad. Sci. (1988), 85:3995. However, unfortunately, the resulting antibodies were typically of the IgM and not the IgG sub class McCabe et al, Cancer Research (1988), 48, 4348-4353, Koda et al, Hum. Antibod. Hybridomas, (1990), 1:15 and secondary (IgG) responses have only been reported with protocols using lymphocytes from immunized donors. Therefore, it would appear that these protocols only succeed in inducing a primary immune response but require donor immunized cells for generation of recall responses.
Also, research has been conducted to systematically analyze cultivation and immunization variables to develop a general protocol for effectively inducing human monoclonal antibodies in vitro Boerner, J. Immunol. (1991) 147:86-95, Brams et al, Hum. Antibod. Hybridomas (1993), 4, 47-56, Lu et al, Hybridoma (1993), 12, 381-389. This has resulted in the isolation of human monoclonal antibodies specific for ferritin Boerner et al, J. Immunol. (1991), 147:86-95, induced by IVI of naive human spleen cells. Also, this research has resulted in a protocol by which de novo secondary (IgG) responses may be induced entirely in vitro Brams et al, Hum. Antibod. Hybridomas (1993), 4, 57-65.
However, despite the great potential advantages of IVI, the efficiency of such methods are severely restricted because of the fact that immune cells grow in monolayers in culture vessels. By contrast, in vivo germinal centers possessing a three-dimensional structure are found in the spleen during the active phases of an immune response. These three-dimensional structures comprise activated T- and B-cells surrounded by antigen-presenting cells which are believed by the majority of immunologists to compare the site of antigen-specific activation of B-cells.
An alternative to the natural splenic environment is to xe2x80x9crecreatexe2x80x9d or mimic splenic conditions in an immunocompromised animal host, such as the xe2x80x9cSevere Combined Immune Deficientxe2x80x9d (SCID) mouse. Human lymphocytes are readily adopted by the SCID mouse (hu-SCID) and produce high levels of immunoglobulins Mosier et al, Nature (1988), 335:256, McCune et al, L. Science (1988), 241, 1632-1639. Moreover, if the donor used for reconstitution has been exposed to a particular antigen, a strong secondary response to the same antigen can be elicited in such mice. For example, Duchosal et al, Duchosal et al, Nature (1992), 355:258-262 reported that human peripheral blood B-cells from a donor vaccinated with tetanus toxoid 17 years prior could be restimulated in the SCID environment to produce high serum levels, i.e., around 104. They further disclosed cloning and expression of the genes of two human anti-TT antibodies using the lambda and the M13 phage combinatorial library approach Huse et al, R.A. Science (1989), 246:1275 from the extracted human cells. The reported antigen affinities of the antibodies were in the 10xc2x0-10xc2x0/M range. However, this protocol required donor primed cells and the yield was very low, only 2 clones were obtained from a library of 370,000 clones.
Therefore, previously the hu-SPL-SCID mouse has only been utilized for producing human monoclonal antibodies to antigens wherein the donor has either been efficiently primed naturally or by vaccination Stxc3xa4hli et al, Methods in Enzymololy (1983), 92, 26-36, which in most cases involves exposure to viral or bacterial antigens. Also, the reported serum titer levels using the hu-SCID animal model are significantly lower than what is typically achieved by immunization of normal mice.
Additionally, two protocols have been described by which induction of primary antibody responses can be followed by induction of secondary antibody responses in hu-SCID mice using naive human lymphocytes. However, use of both of these protocols are substantially restricted. In the first protocol, primary responses are induced in hu-SCID mice into which human fetal liver, thymus and lymph nodes have been surgically implanted. However, this method is severely restricted by the limited availability of fetal tissue, as well as the complicated surgical methodology of the protocol McCune et al, L. Science (1988), 241, 1632-1639. In the second protocol, lethally irradiated normal mice were reconstituted with T- and B-cell depicted human bone marrow and SCID mouse bone marrow cells Lubin et al, Science, (1991), 252:427. However, this method is disadvantageous because it requires a four month incubation period. Moreover, both protocols result in very low antibody titers, i.e., below 104.
Also, Carlson et al, Carlsson et al, J. Immunol. (1992), 148:1065-1071 described in 1992 an approach using PBMCs from an antigen (tetanus toxoid) primed donor. The cells were first depleted of macrophages and NK cells before being subjected to a brief in vitro cultivation and priming period prior to transfer into a SCID mouse. The hu-SPL-SCID mouse was then boosted with antigen. This method was reported to result in average TT specific human IgG titers of ≅104 in the hu-SPL-SCID serum, with up to 5xc3x97105 reported.
Production of human monoclonal antibodies further typically requires the production of immortalized B-cells, in order to obtain cells which secrete a constant, ideally permanent supply of the desired human monoclonal antibodies. Immortalization of B-cells is generally effected by one of four approaches: (i) transformation with EBV, (ii) mouse-human heterofusion, (iii) EBV transformation followed by heterofusion, and (iv) combinatorial immunoglobulin gene library techniques.
EBV transformation has been used successfully in a number of reports, mainly for the generation of anti-HIV antibodies Gornv. et al, Proc. Natl. Acad. Sci. (1989), 86:1624-1628, Posner et al J. Immunol. (1991), 146:4325-32. The main advantage is that approximately one of every 200 B-cells becomes transformed. However, EBV transformed cells are typically unstable, produce low amounts of mainly IgM antibody, clone poorly and cease making antibody after several months of culturing. Heterofusion Carrol, et al, J. Immunol. Meth. (1986), 89:61-72 is typically favored for producing hybridomas which secrete high levels of IgG antibody. Hybridomas are also easy to clone by limiting dilution. However, a disadvantage is the poor yield, i.e., xe2x89xa61 hybridomas per 20,000 lymphocytes Boerner, et al, J. Immunol. (1991), 147:86-95, Ohlin. et al, C.A.K. Immunology (1989), 68:325, Xiu-mei et al, Hum. Antibod. Hybridomas (1990), 1:42, Borrebaeck C.A.K. Abstract at the xe2x80x9cSecond International Conference xe2x80x9d on xe2x80x9cHuman Antibodies and Hybridomas,xe2x80x9d Apr. 26-28, 1992, Cambridge, England. Combining EBV transformation followed by heterofusion offers two advantages: (i) human B-cells fuse more readily to the fusion partner after EBV transformation, and (ii) result in more stable, higher producing hybridomas Ohlin, et al, Immunology (1989), 68:325, Xiu-mei. et al, Hum. Antibod. Hybridomas (1990), 1:42, Borrebaeck C.A.K. Absract at the xe2x80x9cSecond International Conferencexe2x80x9d on xe2x80x9cHuman Antibodies and Hybridomas,xe2x80x9d Apr. 26-28, 1992, Cambridge, England. The advantage of the final technique, i.e., combinatorial immunoglobulin gene library technique is the fact that very large libraries can be screened by means of the M13 Fab expression technology Huse, et al, Science (1989), 246:1275, William Huse. Antibody Engineering: A Practical Guide. Borrebaeck C.A.K. ed. 5:103-120 and that the genes can easily be transferred to a production cell line. However, the yield is typically extremely low, on the order of 1 per 370,000 clones Duchosal, et al, Nature (1992), 355:258-262.
Thus, based on the foregoing, it is apparent that more efficient methods for producing human monoclonal antibodies, in particular antibodies specific to RSV, would be highly advantageous. Moreover, it is also apparent that human antibodies specific to the RSV F-protein having superior binding affinity, specificity and effector functions than those currently available would also be highly desirable.
It is an object of the invention to provide improved methods for producing human antibodies of high titers which are specific to desired antigens.
It is a more specific object of the invention to provide a novel method for producing high titer human antibodies which comprises (i) antigen priming of naive human splenocytes in vitro, (ii) transferral of in vitro antigen primed splenocyte cells to an immunocompromised donor, e.g., a SCID mouse, and (iii) boosting with antigen.
It is another specific object of the invention to provide improved methods for producing human monoclonal antibodies which are specific to respiratory syncytial virus (RSV), and in particular the RSV fusion (F) protein.
It is another object of the invention to provide an improved method for producing EBV immortalized B-cells which favors the formation of EBV immortalized B-cells which predominantly secrete IgG.
It is a more specific object of the invention to provide an improved method for producing EBV immortalized human B-cells which predominantly secrete IgG""s which comprises:
(i) antigen priming of naive human splenocytes in vitro;
(ii) transferral of such in vitro antigen primed naive splenocytes to an immunocompromised donor, e.g., a SCID mouse;
(iii) boosting the immunocompromised donor with antigen;
(iv) isolation of human antibody producing B-cells from the antigen boosted immunocompromised donor, e.g., SCID mouse; and
(v) EBV transformation of said isolated human antibody producing B-cells.
It is another object of the invention to provide novel compositions containing EBV transformed human B-cells obtained from SCID mice which predominantly secrete human IgG""s.
It is a more specific object of the invention to provide novel compositions containing EBV transformed human B-cells which predominantly secrete human IgG""s produced by a method comprising:
(i) antigen priming of naive human splenocytes in vitro;
(ii) transferral of resulting in vitro antigen primed naive splenocytes to an immunocompromised animal donor, e.g., a SCID mouse;
(iii) boosting the immunocompromised animal donor, e.g., SCID mouse, with antigen;
(iv) isolation of human antibody producing B-cells from the antigen boosted immunocompromised donor, e.g., SCID mouse; and
(v) EBV transformation of said isolated human antibody producing B-cells.
It is another specific object of the invention to produce RSV neutralizing human monoclonal antibodies having an affinity to the RSV F-protein of xe2x89xa62xc3x9710xe2x88x929 Molar.
It is still another object of the invention to provide EBV immortalized cell lines which secrete RSV neutralizing human IgG monoclonal antibodies having an affinity to the RSV F antigen of xe2x89xa62xc3x97109 Molar.
It is a more specific object of the present invention to provide two EBV immortalized cell lines, RF-2 and RF-1, which respectively secrete human monoclonal antibodies also referred to as RF-2 and RF-1 which neutralize RSV in vivo and each possess an affinity for the RSV F-protein of xe2x89xa62xc3x9710xe2x88x929.
It is another object of the invention to transfect eukaryotic cells with DNA sequences encoding the RF-1 or RF-2 heavy and light variable domains to produce transfectants which secrete human antibodies containing the variable domain of RF-1 or RF-2.
It is a more specific object of the invention to provide transfected CHO cells which express the RF-1 or RF-2 heavy and light variable domains.
It is another object of the invention to treat or prevent RSV infection in humans by administering a therapeutically or prophylactically effective amount of RSV neutralizing human monoclonal antibodies which are specific to the RSV F-protein and which exhibit a Kd for the RSV F-protein of xe2x89xa62xc3x9710xe2x88x929 molar.
It is a more specific object of the invention to treat or prevent RSV infection in humans by administering a therapeutically or prophylactically effective amount of RF-1 or RF-2 or a human monoclonal antibody expressed in a transfected eukaryotic cell which contains and expresses the variable heavy and light domains of RF-1 or RF-2.
It is another object of the invention to provide vaccines for treating or preventing RSV infection which comprise a therapeutically or prophylactically effective amount of human monoclonal antibodies specific to the RSV F-protein having a Kd for the RSV F-protein of xe2x89xa62xc3x9710xe2x88x929 molar, which neutralize RSV in vitro, in combination with a pharmaceutically acceptable carrier or excipient.
It is a more specific object of the invention to provide vaccines for treating or preventing RSV infection which comprise a therapeutically or prophylactically effective amount of RF-1 or RF-2 or human monoclonal antibodies derived from a transfected eukaryotic cell which contains and expresses DNA sequences encoding the variable heavy and light domains of RF-1 or RF-2, in combination with a pharmaceutically acceptable carrier or excipient.
It is another object of the present invention to provide a method for diagnosis of RSV infection by assaying the presence of RSV in analytes, e.g., respiratory fluids using human monoclonal antibodies which possess an affinity for the RSV fusion (F) protein or xe2x89xa62xc3x9710xe2x88x929 molar.
It is still another object of the invention to provide novel immunoprobes and test kits for detection of RSV infection which comprise human monoclonal antibodies specific to the RSV F-protein, which possess an affinity for the RSV F protein of xe2x89xa62xc3x9710xe2x88x929 molar, which antibodies are directly or indirectly attached to a suitable reporter molecule, e.g., an enzyme or a radionuclide. In the preferred embodiment these human monoclonal antibodies will comprise RF-1 or RF-2 or recombinant human monoclonal antibodies produced in eukaryotic cells, e.g., CHO cells, which are transfected with the variable heavy and light domains of RF-1 or RF-2.
The present invention in its broadest embodiments relates to novel methods for making human antibodies to desired antigens, preferably antigens involved in prophylaxis, treatment or detection of a human disease condition. These methods comprise antigen priming of native human splenocytes in vitro, transferral of the resultant in vitro antigen primed splenocyte cells to an immunocompromised donor, e.g., a SCID mouse, and boosting said immunocompromised donor with antigen.
The present invention also relates to methods for producing Epstein-Barr Virus (EBV) immortalized B-cells which favors the production of cells which secrete IgGs comprising: antigen priming of naive human splenocytes in vitro; transferral of resultant in vitro antigen primed splenocytes to an immunocompromised donor, e.g., a SCID mouse; boosting the immunocompromised donor with antigen; isolating human antibody secreting B-cells, preferably IgG secreting, from the antigen boosted immunocompromised donor, e.g., SCID mouse; and EBV transformation of said isolated human antibody secreting cells.
The present invention more specifically relates to improved methods for making human antibodies to RSV, in particular the RSV fusion (F) protein which exhibit high affinity to RSV F-protein and which also neutralize RSV infection, as well as the human monoclonal antibodies which result from these methods. This is preferably effected by priming of naive human splenocytes in vitro with I1-2 and optionally the RSV F-protein; transferral of the resultant in vitro primed splenocyte cells to an immunocompromised donor, e.g., a SCID mouse, and boosting with RSV F-protein to produce human B-cells which secrete neutralizing anti-RSV F-protein human antibodies having high affinity to the RSV F-protein, i.e., xe2x89xa62xc3x9710xe2x88x929 molar.
The resultant B-cells are preferably immortalized so as to provide a constant stable supply of human anti-RSV F-protein monoclonal antibodies. In the preferred embodiment B-cells are isolated from the antigen boosted SCID mouse and transformed with EBV virus to produce EBV transformed human B-cells which predominantly secrete human IgGs.
These cells are then cloned to select EBV transformed cell lines which secrete human monoclonal antibodies having high affinity to RSV F-protein, i.e. xe2x89xa610xe2x88x927 and preferably xe2x89xa62xc3x9710xe2x88x929 molar.
The present invention also relates to the use of such anti-RSV F-protein human monoclonal antibodies as therapeutic and/or prophylactic, as well as diagnostic agents. As noted, the subject methods result in the generation of human monoclonal antibodies which exhibit high affinity to the RSV F-protein, i.e., which possess a Kd for the RSV F-protein of xe2x89xa62xc3x9710xe2x88x929 molar, which also neutralize RSV in vitro. Therefore, these antibodies are ideally suited as prophylactic and therapeutic agents for preventing or treating RSV infection given the fact that the RSV F-protein is a surface protein which is highly conserved across different RSV isolates. Also, given the high affinity and specificity of the subject human monoclonal antibodies to RSV F-protein, they also may be used to diagnose RSV infection.
More specifically, the present invention provides two particular human monoclonal antibodies to the RSV F-protein, i.e., RF-1 and RF-2, as well as recombinant human antibodies derived therefrom, which are preferably produced in CHO cells, which cells have been transfected with DNA sequences encoding the variable heavy and light domains of RF-1 or RF-2. These antibodies are particularly useful as prophylactic and/or therapeutic agents for treatment or prevention of RSV infection. Moreover, these antibodies are useful as diagnostic agents because they bind the RSV F-protein with high affinity, i.e., each possess affinity for the RSV F-protein of xe2x89xa62xc3x9710xe2x88x929. They are especially useful as therapeutic agents because of their high affinity and specificity for the RSV F-protein, and their ability to effectively neutralize RSV infection in vitro.